Genetic association and transcriptome integration identify contributing genes and tissues at cystic fibrosis modifier loci

Abstract

Cystic Fibrosis (CF) exhibits morbidity in several organs, including progressive lung disease in all patients and intestinal obstruction at birth (meconium ileus) in ~15%. Individuals with the same causal CFTR mutations show variable disease presentation which is partly attributed to modifier genes. With >6,500 participants from the International CF Gene Modifier Consortium, genome-wide association investigation identified a new modifier locus for meconium ileus encompassing ATP12A on chromosome 13 (min p = 3.83x10(-10)); replicated loci encompassing SLC6A14 on chromosome X and SLC26A9 on chromosome 1, (min p<2.2x10(-16), 2.81x10(-11), respectively); and replicated a suggestive locus on chromosome 7 near PRSS1 (min p = 2.55x10(-7)). PRSS1 is exclusively expressed in the exocrine pancreas and was previously associated with non-CF pancreatitis with functional characterization demonstrating impact on PRSS1 gene expression. We thus asked whether the other meconium ileus modifier loci impact gene expression and in which organ. We developed and applied a colocalization framework called the Simple Sum (SS) that integrates regulatory and genetic association information, and also contrasts colocalization evidence across tissues or genes. The associated modifier loci colocalized with expression quantitative trait loci (eQTLs) for ATP12A (p = 3.35x10(-8)), SLC6A14 (p = 1.12x10(-10)) and SLC26A9 (p = 4.48x10(-5)) in the pancreas, even though meconium ileus manifests in the intestine. The meconium ileus susceptibility locus on chromosome X appeared shifted in location from a previously identified locus for CF lung disease severity. Using the SS we integrated the lung disease association locus with eQTLs from nasal epithelia of 63 CF participants and demonstrated evidence of colocalization with airway-specific regulation of SLC6A14 (p = 2.3x10(-4)). Cystic Fibrosis is realizing the promise of personalized medicine, and identification of the contributing organ and understanding of tissue specificity for a gene modifier is essential for the next phase of personalizing therapeutic strategies.

Fig 1. Manhattan plot for the genome-wide association study with meconium ileus in the GMC sample of 6,770 individuals with CF.

All variants with MAF>1% are included in the analysis. The solid horizontal line corresponds to the genome-wide significance threshold of p = 5x10^-8 [30], and the dotted horizontal line is the false discovery rate control threshold of q = 0.05 (equivalent to p<7.67x10^-7) [31]_. Accuracy of p-value calculation was up to 2.2x10^-16 by the geeglm function [100] in R. Variants rs4077468 and rs7512462 identified in previous CF gene modifier studies also noted.

Fig 2. Overlay of the p-values of individual SNPs from the meconium ileus GWAS and GTEx eQTL association and the enhancer and promoter states from the REMC at the three genome-wide significant loci.

All the p-values are on the–log₁₀ scale with the left y-axis representing the GWAS (with the filled diamond for the SNP with the lowest p-value and colored dots indicating the LD with this SNP) and the right y-axis representing the GTEx eQTL results (version 7; with colored lines indicated for different tissues) for SLC6A14 (A), SLC26A9 (B), and ATP12A (C). Note that eQTLs for SLC6A14 in the pancreas and ATP12A in the transverse colon were not mapped by GTEx v7, but were calculated and included here—refer to Material and Methods, GTEx Data section for details. To display the eQTL data, each physical region shown was divided into 25 equal windows and lines were drawn for each tissue by connecting the minimum GTEx eQTL p-value within each window for that gene. The absence of a line indicates insufficient gene expression for that particular tissue (see GTEx Data section for details on expression threshold). In the lower panels, the 5-mark 15-state observed chromHMM enhancer (orange) and promoter (red) states within DNase hypersensitive sites are shown (Material and Methods) from adult pancreas (Panc), fetal small intestine (FIS) and adult small intestine (SI).

Fig 3. SLC6A14 meconium ileus and lung association and gene expression profiles point to cis-regulation that is distinct in pancreas and lung.

(A) Overlay of p-values (on the–log₁₀ scale) from the meconium ileus GWAS (red/yellow palette of colored dots; this study), from the previous lung function GWAS (green/blue palette of colored dots; [41]) and from the GTEx eQTL for association between SNPs and SLC6A14 expression (lines, derived as for Fig 2) for tissues of interest. (B) and (C) Slc6a14 gene expression levels measured by RT-PCR of RNA from C57BL6/J mice (n = 4) from pancreas (B) and lung (C); expression is shown relative to Gusb at gestational (E) or post-natal (P) days indicated.